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===Bisulfite Conversion of Tagged Nextera Fragments=== *Add 1.172ul 50ng ssDNA to each tube as a carrier (42.68ng/ul) * Add 11.83ul ddh2o to all tubes make it up to 20ul Prepare separate tube of 1ng DNA go through bisulfite conversion with 95C 5m denaturing step 5.68ul DNA with 14.32ul h2o *Add in 130uL of CT conversion Reagent Solution directly to the digested samples *Vortex and spin down *Perform reaction in thermocycler Step1 98C, 8m Step2 64C, 3.5hr Step4 4C, storage for up to 20 hr *Add 600uL of M-Bindin Buffer into a IC Column *Load 150uL of samples into IC column '''CLOSE CAP AND MIX BY INVERTING THE COLUMN SEVERAL TIMES''' *Centrifuge at 14,000rpm for 30s *Discard flow through *Add 200uL of M-Desulphonation Buffer to column let stand at RT for 14m *Repeat centrifuge step *Add 200uL of M-Wash Buffer to the column *Repeat Centrifuge *Place column in a 1.5mL tube *Add in 11uL of warm M-Elution Buffer (37C) directly to the column matix, sit at 37C for 5 min *Repeat Centrifuge
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